Synthesis and structure-activity relationship of thiobarbituric acid derivatives as potent inhibitors of urease

Article abstract of DOI:10.1016/j.bmc.2014.05.057

A series of thiobarbituric acid derivatives 1-27 were synthesized and evaluated for their urease inhibitory potential. Exciting results were obtained from the screening of these compounds 1-27. Compounds 5, 7, 8, 11, 16, 17, 22, 23 and 24 showed excellent urease inhibition with IC₅₀ values 18.1 ± 0.52, 16.0 ± 0.45, 16.0 ± 0.22, 14.3 ± 0.27, 6.7 ± 0.27, 10.6 ± 0.17, 19.2 ± 0.29, 18.2 ± 0.76 and 1.61 ± 0.18 μM, respectively, much better than the standard urease inhibitor thiourea (IC₅₀ = 21 ± 0.11 μM). Compound 3, 4, 10, and 26 exhibited comparable activities to the standard with IC₅₀ values 21.4 ± 1.04 and 21.5 ± 0.61μM, 22.8 ± 0.32, 25.2 ± 0.63, respectively. However the remaining compounds also showed prominent inhibitory potential The structure-activity relationship was established for these compounds. This study identified a novel class of urease inhibitors. The structures of all compounds were confirmed through spectroscopic techniques such as EI-MS and ¹H NMR.

Full text of DOI:10.1016/j.bmc.2014.05.057

Accepted Manuscript
Synthesis and Structure-activity Relationship of Thiobarbituric Acid Deriva-
tives as Potent Inhibitors of Urease
Khalid Mohammed Khan, Fazal Rahim, Ajmal Khan, Muhammad Shabeer,
Shafqat Hussain, Wajid Rehman, Muhammad Taha, Momin Khan, Shahnaz
Perveen, M. Iqbal Choudhary
PII:
S0968-0896(14)00416-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.05.057
BMC 11618
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 April 2014
22 May 2014
23 May 2014
Please cite this article as: Khan, K.M., Rahim, F., Khan, A., Shabeer, M., Hussain, S., Rehman, W., Taha, M., Khan,
M., Perveen, S., Iqbal Choudhary, M., Synthesis and Structure-activity Relationship of Thiobarbituric Acid
Derivatives as Potent Inhibitors of Urease, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/
10.1016/j.bmc.2014.05.057
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis and Structure-activity Relationship of Thiobarbituric Acid Derivatives as
Potent Inhibitors of Urease
Khalid Mohammed Khan,1∗ Fazal Rahim,2 Ajmal Khan,1 Muhammad Shabeer,2 Shafqat
Hussain,1 Wajid Rehman,2 Muhammad Taha,3,4 Momin Khan,6 Shahnaz Perveen,5 and M.
Iqbal Choudhary1
1H. E. J. Research Institute of Chemistry, International Center for Chemical and
Biological Sciences, University of Karachi, Karachi-75270, Pakistan
2Depatment of Chemistry, Hazara University, Mansehra, Pakistan
3Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA
(UiTM), Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor, Malaysia,
4Faculty of Applied Science UiTM, 40450 Shah Alam, Selangor, Malaysia,
5PCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi
75280, Pakistan
6Department of Chemistry, Abdul Wali Khan University Mardan, Mardan-23200,
Pakistan.
Abstract
A series of thiobarbituric acid derivatives 1-27 were synthesized and evaluated for their
urease inhibitory potential. Exciting results were obtained from the screening of these
compounds 1-27. Compounds 5, 7, 8, 11, 16, 17, 22, 23 and 24 showed excellent urease
inhibition with IC50 values 18.1 0.52, 16.0 0.45, 16.0 0.22, 14.3 0.27, 6.7 0.27,
10.6 0.17, 19.2 0.29, 18.2 0.76 and 1.61 0.18 µM, respectively, much better than the
standard urease inhibitor thiourea (IC50 = 21
0.11 µM). Compound 3, 4, 10, and 26
exhibited comparable activities to the standard with IC50 values 21.4 1.04 and 21.5
*Corresponding Author: khalid.khan@iccs.edu, hassaan2@super.net.pk, Tel.: 0092-21-34824910; Fax:
0092-21-34819018
1

0.61µM, 22.8 0.32, 25.2 0.63, respectively. However the remaining compounds also
showed prominent inhibitory potential The structure-activity relationship was established
for these compounds. This study identified a novel class of urease inhibitors. The
structures of all compounds were confirmed through spectroscopic techniques such as EI-
MS and 1HNMR.
Keywords: Thiobarbituric acid analogs, Synthesis, Urease Inhibition, SAR Studies.
1.
Introduction
Urease enzyme is a virulence factor in certain human and animal ailments. It contributes
to the development of kidney stones, pyelonephritis, peptic ulcers leading to gastric
cancers and other diseases.1 It also causes the pathogenesis of pyelonephritis, hepatic
encephalopathy, hepatic coma urolithiasis, ammonia and urinary catheter encrustation.2
The obvious cure for treating bacterial infection with antimicrobials, however, often
4-5
proved to be unsuccessful.3 Currently it was reported that the gastric cancer
is the
fourth most common cancer and the second most common cause of cancer-related deaths
worldwide.6 It is also a cause of pathologies due to Helicobacter pylori, which lets
bacteria to persist at the low pH of the stomach during colonization lead pathogenesis of
gastric and peptic ulcer which in the long run may cause cancer.7
The barbiturates and thiobarbiturates showed a wide range of pharmacological
applications, such as general anesthesia, sedation and anticonvulsant and anxiolytic
effects. Barbiturate compounds also showed urease inhibition.8 Barbituric and
9-10
11
antifungal, antiviral,
12
thiobarbituric acid derivatives also exhibited antimicrobial,
and antitumor activities.13 From literature study it is clear that barbiturates and
thiobarbiturate derivatives show diverse biological activities such as potential mushroom
14
tyrosinase inhibition, antituberculosos,
15
16
radio-sensitization, anticancer with anti-
17
18
inflammatory activities,
inhibition for diaminopimelate aminotransferase,
and
anesthesia.19 Barbiturate analogues also showed anti-proliferative activity.20
Our research group is involved in the search for simple but biologically interesting
molecules that are easy to synthesize with no tedious chemistry and that could be
accomplished in just fewer steps with high yields. This type of chemistry is easily and
ideally adopted by the pharmaceutical industry for commercialization. Previously, our
2

research group reported arylidene barbiturates as urease inhibitors their other activities.21
In view of these studies; we planned to synthesize thiobarbiturate derivatives with the
aim to discover their urease inhibition.
1. Results and discussion
2.1 Chemistry
The synthesis of thiobarbituric acid derivatives (1-27) was carried out by the reaction of
thiobarbituric acid (1 mmol) with different aromatic aldehyde (1 mmol) in the presence
of 10 ml of 20% NaOH. The reaction mixture was stirred for 12 h. The completion of
reaction was monitored by TLC. After completion of the reaction, the mixture was
poured onto crushed ice followed by acidification with dilute hydrochloric acid. The
products (1-27) were precipitated out; the solid was filtered, dried and recrystallized from
ethanol (Scheme-1, Table-1). Structures of synthetic compounds were identified by
1
spectroscopic methods such as H NMR and EIMS. All synthetic derivatives furnished
satisfactory elemental analyses.
Insert Scheme 1 here
Insert Table 1 here
2.2 Urease Inhibitory Activity
Compounds 1-27 were evaluated for inhibition of urease enzyme. All compounds showed
potent urease inhibitory activity (Table-2). Compounds 5, 7, 8, 11, 16, 17, 22, 23 and 24
showed excellent urease inhibition with IC50 values 18.1 0.52, 16.0 0.45, 16.0 0.22,
14.3
respectively, much better than the standard inhibitor thiourea (IC50 = 21
Compound 3, 4, 10, and 26 exhibited comparable activities to the standard with IC50 values
21.4 1.04 and 21.5 0.61µM, 22.8 0.32, 25.2 0.63, respectively . However,
0.27, 6.7
0.27, 10.6
0.17, 19.2
0.29, 18.2
0.76 and 1.61 0.18 µM,
0.11 µM).
compounds 1, 2, 6, 9, 12, 14, 15, 18, 19, 20, 21, and 25 also exhibited striking inhibitory
potential with IC50 values 46.5 0.56, 47.3 0.62, 62.8 1.71, 43.3 1.06, 42.5 1.3, 50.3
0.81, 32.3 0.62, 58.5 1.28, 42.1 1.91, 29.4 0.59, 32.1 0.34, and 32.7 0.82 µM,
respectively.
3

Structure-activity relationship suggested that the urease activity of a particular molecule
is apparently governed by the substitution present at aromatic residues. The p-thiomethyl
substituted analog 5 (IC50 = 18.1 0.52 µM), pyridin-4-yl 7 (IC50 = 16.0 0.45 µM), 2-
methyl-o-pyridin-2-yl 8 (IC50 = 16.0 0.22 µM), 3,4-dimetoxy 11 (IC50 = 14.3 0.27
µM), p-phthalaldehydic 16 (IC50 = 6.7 0.27 µM), 3,5-dibromo-4-hyroxy 17 (IC50 = 10.6
0.17 µM), 2-hydroxy-3-methoxy 22 (IC50 = 19.2 0.29 µM), 2-methylfuryl 23 (IC50 =
18.2 0.76 µM), and 3,4-dihydroxy 24 (IC50 = 1.61 0.18 µM), respectively, showed
excellent inhibitory activities among the series. It was observed that variation in
substitution pattern of benzaldehyde or aromatics resulted a difference in activities.
Compound 24 (3, 4-dihydroxy analog) is found to be the most active among the series
with IC50 value 1.61 0.18 µM. The two hydroxyl groups on phenyl ring might be
involved in hydrogen bonding with nickel atoms present in urease enzyme. The
compound 16 got second position among the series with IC50 value 6.7 0.27 µM. This
compound has one more sulfur group which might help in coordination with the nickels.
The compounds 17 and 22 having p-hydroxy and o-hydroxy group with IC50 values 10.6
0.17 and 19.2
0.29 µM, respectively, also showed potent inhibition. The slight
activity difference is due to differences in position of hydroxyl groups on the phenyl ring.
The p-pyridyl 7 and 2-methyl-o-pyridyl 8 have almost identical urease inhibition with
IC50 values 16.0 0.45, 16.0 0.22 µM. The excellent inhibitory potential of these two
compounds may be due to the lone pair interaction of pyridines nitrogens with nickels.
The p-thiomethyl substituted 5 and 3,4-dimetoxy 11 having IC50 values of 18.1 + 0.52
and 14.3 + 0.27 µM, respectively, have substituents with electron donating inductive
effect. The 2-methylfuryl analog 23 have IC50 value 18.2 0.76 µM displayed greater
potential that may be due to lone pair interaction of furfuryl oxygen with nickel atom.
The 2-naphthol residue 3 and thiophenyl analog 4 displayed IC50 values 21.4 1.04 and
21.5
0.61 µM, respectively, were found to have comparable IC50 values with the
standard. The 4-hydroxy-3,5-dimethoxy substituted analog 1, 3-bromo-4,5-dimethoxy 2,
coumaryl 6, 4-bromo-2,5-dimethoxy 9, 2-hydroxy-4-methoxy 10, 4-hydroxy-3-iodo-5-
methoxy 12, 3,5-dichloro-2-hydroxy 14, 2-amino 15, N,N-dimethylamino 18, 2-methyl
19, p-ethoxy 20, 2,4-dihydroxy 21, 2-hydroxy-5-methoxy 25 and 2,3,4-trihydroxy 26
showed good to excellent inhibition. The dimethoxy analogs i.e. 4-hydroxy-3,5-
4

dimethoxy substituted analog 1, 3-bromo-4,5-dimethoxy analog 2, and 4-bromo-2,5-
dimethoxy analog 9, having IC50 values 46.5 0.56, 47.3 0.62 and 43.3 1.06 µM,
respectively, showed almost good potential. The change in position of substituents
creates a difference of activities in smaller magnitude. The compounds having both
methoxy and hydroxyl like in 2-hydroxy-4-methoxy analog 10, 4-hydroxy-3-iodo-5-
methoxy analog 12 and 2-hydroxy-5-methoxy analog 25 showed excellent inhibition. The
compounds 10 and 25 having same substituents but the position of substituents are
different that brings a difference in their activities. By comparing the activities of mono,
di and tri hydroxyl analogs like 3, 5-dichloro-2-hydroxy 14, 2,4-dihydroxy 21 and 2,3,4-
trihydroxy 26, we found that urease inhibitory potential increases with the increase in the
number of hydroxyl groups on aromatic ring. By changing the substituent from hydroxyl
to amino group such as 2-amino 15 and N,N-dimethylamino 18 it was observed that the
unsubstituted amino analog showed greater potential than the substituted one which
might be due to the obvious reason of better interaction of free nitrogen with nickel atom.
Comparing the inhibitory potential of compound 2-methyl 19 and p-ethoxy analog 20, we
observed that as the electron donating power of a group increases, the inhibitory potential
proportionally increases. The coumaryl 6, anthranyl 13 and the indolyl 27 showed weak
inhibition with IC50 values of 62.8 1.71, 137.2 1.92 and 170.8 2.08µM, respectively.
All these compounds showed weak inhibition and this might be due to the steric
hindrance.
Insert table 2 here
2. Conclusion
This study guided to the bioorganic and medicinal chemist that a simple one step
chemistry may generate extra-ordinary bioactive compounds. During this study, herein,
we synthesized twenty-seven thiobarbituric acid derivatives through a simple one step
chemistry and evaluated for their inhibitory potential against urease. Most of these
compounds were identified as excellent urease inhibitors. This study discovered a new
class of urease inhibitors. These lead compounds have opened a new avenue that has the
potential to be novel inhibitors against urease through further modifications.
5

3. Material and methods
1H NMR spectra were recorded in DMSO-d6 on an Avance Bruker AM 300-500 MHz
instruments and TMS was used as an external standard. Chemical shifts are given in δ
(ppm).
Electron impact mass spectra (EI MS) were recorded on a Finnigan MAT-311A,
Germany. CHN Analyses were carried out a Carlo Erba Strumentazion-Mod-1106, Italy.
Thin layer chromatography (TLC) was performed on pre-coated silica gel aluminum
plates (Kieselgel 60, 254, E. Merck, Germany). Chromatograms were visualized by UV
at 254 and 365 nm.
4.1 General procedure for the synthesis of compounds 1-27
The syntheses of thiobarbituric acid derivatives (1-27) were carried out by the reaction of
thiobarbituric acid (1 mmol) with different aromatic aldehyde (1 mmol) in the presence
of 10 ml of 20% NaOH. The reaction mixture was stirred for 12 h. The completion of
reaction was monitored by periodic TLC. The completion of the reaction was monitored
periodically by TLC. After completion of the reaction, the mixture was poured into
crushed ice followed by acidification with dilute HCl. The product precipitated as a solid
which was filtered, dried and recrystallized from ethanol.
4.1.1. 5-(4-Hydroxy-3,5-dimethoxybenzylidene)-2-thioxodihydro-4,6(1H,5H)-
pyrimidinedione (1)
°
1
Yield: 0.88 g (93%); MP 263 C: H-NMR: (DMSO-d6, 300 MHz): δ 12.3 (s, 2H, NH),
11.1 (s, 1H, OH), 8.3 (s, 1H, CH aldehydic), 7.3 (s, 2H, H-2/6), 3.7 (s, 6H, OCH3); EI-
MS: m/z (rel. int. %): 308 (M+, 100), 280 (69), 224 (44), 190 (35), 165 (30).
4.1.2 5-(2-Bromo-4,5-dimethoxybenzylidene)-2-thioxodihydro-4,6(1H,5H)-
pyrimidinedione (2)
°
1
Yield: 0.89 g (92%); MP 271 C: H-NMR: (DMSO-d6, 300 MHz): δ 12.4 (s, 2H, NH),
8.5 (s, 1H, H-3), 8.2 (s, 1H, CH aldehydic), 7.9 (s, 1H, H-6), 3.9 (s, 6H, OCH3); EI-MS:
m/z (rel. int. %): 306 (M+, 90), 304 (90) 277 (93), 260 (100), 247 (29) 179 (43).
6

4.1.3 5-[(2-Hydroxy-1-naphthyl)methylene]-2-thioxodihydro-4,6(1H,5H)-
pyrimidinedione (3)
°
Yield: 0.89 g (93%); MP 255 C:1H-NMR: (DMSO-d6, 300 MHz): δ 12.4 (s, 1H, NH),
12.3(s, 1H, NH),11.2 (s, 1H, OH),7.7 (d, 2H, J5,6/8,7 = 8.2 Hz, H-5/8), 7.5 (d, 1H, J4,3 = 8.0
Hz, H-4), 7.3 (m, 2H, H-6/ CH aldehydic), 7.1 (d, 1H, J3,4 = 8.4 Hz, H-3). EI-MS: m/z
(rel. int. %): 298 (M+, 100), 270 (56), 255 (45), 176 (60), 145 (34).
4.1.4 5-(2-Thienylmethylene)-2-thioxodihydro-4,6(1H,5H)-pyrimidinedione (4)
°
1
Yield: 0.89 g (93%); MP 246 C: H-NMR: (DMSO-d6, 300 MHz): δ 12.4 (s, 1H, NH),
12.3 (s, 1H, NH), 8.4 (s, 1H, CH aldehydic), 8.1 (d, 1H, J4,3 = 3.5 Hz, H-4), 7.7 (d, 1H,
J2,3 = 3.6 Hz, H-2), 7.5 (m, 1H,H-3); EI-MS: m/z (rel. int. %): 238 (M+, 100), 220 (74),
180 (55), 159 (36) 115 (23).
4.1.5. 5-[4-(Methylsulfanyl)benzylidene]-2-thioxodihydro-4,6(1H,5H) pyrimidinedione (5)
°
1
Yield: 0.90 g (93%); MP 262 C: H-NMR: (DMSO-d6, 300 MHz): δ 12.4 (s, 1H, NH),
12.3 (s, 1H, NH), 8.3 (s, 1H, CH aldehydic), 8.2 (d, 2H, J3,2/5,6 = 6 Hz, H-3/5), 7.4 (d, 2H,
J2,3/6,5 = 8.4 Hz, H-2/6), 2.6 (s, 3H, SCH3); EI-MS: m/z (rel. int. %): 278 (M+, 278), 260
(74), 244 (93), 176 (66), 134 (50).
4.1.6. 5-[(6-Bromo-4-chloro-2-oxo-2H-chromen-3-yl)methylene]-2-thioxodihydro
4,6(1H,5H)-pyrimidinedione (6)
°
1
Yield: 0.89 g (93%); MP 258 C: H-NMR: (DMSO-d6, 300 MHz): δ 12.3 (s, 1H, NH),
12.2 (s, 1H, NH), 8.5 (s, 1H, H-5), 8.4 (d, 1H, J7’,8’ = 8.2 Hz, H-7’), 8.2 (d, 1H, J8,7 = 8.2
Hz, H-8), 8.0 (s, 1H, CH aldehydic); EI-MS: m/z (rel. int. %): 413 (M+, 100), 380 (60),
320 (45), 290 (63), 250 (33).
4.1.7 5-(4-Pyridinylmethylene)-2-thioxodihydro-4,6(1H,5H)-pyrimidinedione (7)
°
1
Yield: 0.90 g (95%); MP 283 C: H-NMR: (DMSO-d6, 300 MHz): δ 11.75 (s, 2H, NH),
8.64 (d, 2H, J3,2/5,6 = 6.6 Hz, H-3/5), 7.7 (d, 2H, J2,3/6,5 = 5.7 Hz, H-2/6), 6.2 (s, 1H, CH
aldehydic); EI-MS: m/z (rel. int. %): 233 (M+, 100), 174 (15), 135 (24), 115 (21) 103
(24).
7

4.1.8 5-[(6-Methyl-2-pyridinyl)methylene]-2-thioxodihydro-4,6(1H,5H)-
pyrimidinedione (8)
°
1
Yield: 0.88 g (93%); MP 272 C: H-NMR: (DMSO-d6, 300 MHz): δ 11.7 (s, 2H, NH),
8.3 (m, 1H, H-3) 7.7 (d, 1H, J2,3 = 7.8Hz, H-2),7.6 (d, 1H, J3,4 = 8.1Hz, H-4), 6.1 (s,1H,
CH aldehydic), 1.5 (s, 3H, CH3); EI-MS: m/z (rel. int. %): 247 (M+, 62), 219 (69), 148
(80), 143 (100) 116 (47).
4.1.9 5-(4-Bromo-2,5-dimethoxybenzylidene)-2-thioxodihydro-4,6(1H,5H)-
pyrimidinedione (9)
°
1
Yield: 0.96 g (92%); MP 295 C: H-NMR: (DMSO-d6, 300 MHz): δ 12.4 (s, 1H, NH),
12.3 (s, 1H, NH) 8.4 (s, 1H, H-2), 8.0 (s, 1H, H-5), 7.4 (s, 1H, CH aldehydic) 3.9 (s, 3H,
OCH3), 3.8 (s, 3H, OCH3). EI-MS: m/z (rel. int. %): 370 (M+, 62), 340 (100), 338 (75),
282 (51), 280 (56).
4.1.10. 5-(3-Hydroxy-4-methoxybenzylidene)-2-thioxodihydro-4,6(1H,5H)-
pyrimidinedione (10)
°
1
Yield: 0.89 g (93%); MP 276 C: H-NMR: (DMSO-d6, 300 MHz): δ 12.4 (s, 1H, NH),
12.3 (s, 1H, NH), 9.5 (s, 1H,OH), 8.2 (s, 1H, CH aldehydic), 8.1 (s, 1H, H-2), 7.78 (d,
1H, J5,6 = 6.9 Hz, H-5), 7.72 (dd, 1H, J6,5 = 9.2. J6,2 = 2.1 Hz, H-6), 3.9 (s, 3H, OCH3);
EI-MS: m/z (rel. int. %): 278 (M+, 100), 260 (16), 218 (14), 179 (20), 132 (15).
4.1.11. 5-(3,4-Dimethoxybenzylidene)-2-thioxodihydro-4,6(1H,5H)-pyrimidinedione (11)
°
1
Yield: 0.96 g (92%); MP 268 C: H-NMR: (DMSO-d6, 400 MHz): δ 12.4 (s, 1H, NH),
12.3 (s, 1H, NH), 8.4 (d, 1H, J2,6 = 1.6Hz, H-2), 8.3 (s, 1H, CH aldehydic), 7.9 (m, 1H,
H-6), 7.13 (d, 1H, J5,6 = 8.8 Hz, H-5), 3.9 (s, 3H, OCH3), 3.8 (s, 3H, OCH3); EI-MS: m/z
(rel. int. %): 292 (M+, 100), 277 (34), 261 (61), 232 (18), 202 (40).
4.1.12.
5-(4-Hydroxy-3-iodo-5-methoxybenzylidene)-2-thioxodihydro-4,6(1H,5H)
pyrimidinedione(12)
°
1
Yield: 0.91 g (90%); MP 300 C: H-NMR: (DMSO-d6, 300 MHz): δ 12.3 (s, 2H, NH),
8.6 (s, 1H, CH aldehydic), 8.3 (s, 1H, H-2), 8.2 (s, 1H, H-6), 3.9 (s, 3H, OCH3); EI-MS:
m/z (rel. int. %): 404 (M+, 100), 402 (18), 305 (15), 277 (10) 218 (25).
8

4.1.13. 5-(1,4-Dihydro-9-anthracenylmethylene)-2-thioxodihydro-4,6(1H,5H)-
pyrimidinedione (13)
°
1
Yield: 0.91 g (88%); MP 285 C: H-NMR: (DMSO-d6, 400 MHz): δ 12.6 (s, 1H, NH),
12.2 (s, 1H, NH), 8.9 (s, 1H, H-6), 8.7 (s, 1H, CH aldehydic), 8.2 (d, 2H, J2,3/10,9 = 8.4
Hz, H-2/10), 7.9 (d, 2H, J7,8/5,4 = 8.4 Hz,H-7/5), 7.6 (m, 4H, H-3/4/8/9); EI-MS: m/z (rel.
int. %): 334 (M+, 7), 331 (73), 230 (100), 202 (95), 201 (41).
4.1.14. 5-(3,5-Dichloro-2-hydroxybenzylidene)-2-thioxodihydro-4,6(1H,5H)-
pyrimidinedione (14)
°
1
Yield: 0.88 g (92%); MP 281 C: H-NMR: (DMSO-d6, 400 MHz): δ 12.4 (s, 2H, NH),
11.6 (s, 1H, OH), 7.5 (s, 1H, H-4), 7.09 (s, 1H, H-6), 5.2 (s, 1H, CH aldehydic); EI-MS:
m/z (rel. int. %): 317 (M+, 100), 300 (82), 240 (25), 211 (54), 115 (56).
4.1.15. 5-(2-Aminobenzylidene)-2-thioxodihydro-4,6(1H,5H)-pyrimidinedione (15)
°
1
Yield: 0.89 g (93%); MP 249 C: H-NMR: (DMSO-d6, 300 MHz): δ 12.6 (s, 2H, NH),
8.5 (s, 1H, CH aldehydic), 8.2 (d, 1H, J3,4 = 8.1 Hz, H-3), 7.9 (d, 1H, J6,5 = 3.6 Hz, H-
6),7.6 (m, 2H, H-4/5), 4.1 (s, 2H, NH2); EI-MS: m/z (rel. int. %): 247 (M+, 44), 299 (46),
172 (100), 143 (52) 116 (46).
4.1.16. 5-(4-{[4,6-Dioxo-2-thioxotetrahydro-5(2H)pyrimidinylidene]methyl}-
benzylidene)-2-thioxodihydro-4,6(1H,5H)-pyrimidinedione (16)
°
1
Yield: 0.87 g (91%); MP 284 C: H-NMR: (DMSO-d6, 300 MHz): δ 12.4 (s, 4H, NH),
8.2 (s, 2H, H- CH aldehydic), 7.9 (d, 2H, J2,3/6,5 = 8.4 Hz, H-2/6), 7.8 (d, 2H, J3,2/5,6 = 8.4
Hz, H-3/5); EI-MS: m/z (rel. int. %): 386 (M+, 100), 350 (14), 314 (20), 270 (63) 220
(11).
4.1.17. 5-(3,5-Dibromo-4-hydroxybenzylidene)-2-thioxodihydro-4,6(1H,5H)-
pyrimidinedione (17)
°
1
Yield: 0.85 g (89%); MP 290 C: H-NMR: (DMSO-d6, 300 MHz): δ 12.7 (s, 1H, NH),
12.1 (s, 1H, NH), 8.7 (s, 2H,H-2/6), 8.00 (s, 1H, CH aldehydic); EI-MS: m/z (rel. int. %):
406 (M+, 100), 402 (50), 327 (16), 307 (43), 276 (18), 87 (34).
9

4.1.18. 5-[4(Dimethylamino) benzylidene]-2-thioxodihydro-4,6(1H,5H)-pyrimidinedione
(18)
°
1
Yield: 0.87 g (91%); MP 259 C: H-NMR: (DMSO-d6, 300 MHz): δ 12.4 (s, 2H, NH),
8.5 (s, 1H, CH aldehydic), 8.2 (d, 2H, J2,3/6,5 = 8.1 Hz, H-2/6), 7.9 (d, 2H, J3,2/5,6 = 8.2 Hz,
H-3/5), 3.6 (s, 6H, NMe2); EI-MS: m/z (rel. int. %): 275 (M+, 100), 273 (30), 177 (29),
172 (15) 144 (16).
4.1.19. 5-(2-Methylbenzylidene)-2-thioxodihydro-4,6(1H,5H)-pyrimidinedione (19)
°
1
Yield: 0.89 g (93%); MP 241 C: H-NMR: (DMSO-d6, 300 MHz): δ 12.5 (s, 2H, NH),
8.9 (s, 1H, H-7), 7.9 (d, 2H, J3,4/6,5 = 8.7 Hz, H-3/6), 7.1 (m, 2H, H-4/5),2.4 (s, 3H, CH3);
EI-MS: m/z (rel. int. %): 246 (M+, 100), 203 (14), 188 (20), 159 (63), 103 (11).
4.1.20. 5-(4-Ethoxybenzylidene)-2-thioxodihydro-4,6(1H,5H)-pyrimidinedione (20)
°
1
Yield: 0.93 g (91%); MP 263 C: H-NMR: (DMSO-d6, 300 MHz): δ 12.1 (s, 1H, NH),
12.0 (s, 1H, NH), 8.5(d, 2H, J2,3/6,5 = 9.3Hz, H-2/6), 8.2 (s, 1H, CH aldehydic), 6.9 (d,
2H, J3,2/5,6 = 9 Hz, H-3/5), 4.1 (m, 2H, OCH2), 3.2 (m, 3H, OCH2CH3); EI-MS: m/z (rel.
int. %): 276 (M+, 100), 247 (46), 230 (29), 150 (27) 145 (16).
4.1.21. 5-(2,4-Dihydroxybenzylidene)-2-thioxodihydro-4,6(1H,5H)-pyrimidinedione (21)
°
1
Yield: 0.89 g (93%); MP 256 C: HNMR: (DMSO-d6, 300 MHz): δ 12.4 (s, 1H, NH),
12.3 (s, 1H, NH), 11.2 (s, 1H, OH), 8.5 (d, 1H, J6,5 = 3.6 Hz, H-6), 8.2 (s, 1H, CH
aldehydic), 7.97 (s, 1H, H-3), 6.7 (d, 1H, J5,6 = 3.6 HZ, H-5); EI-MS: m/z (rel. int. %):
264 (M+, 100), 243 (10), 208 (40), 203 (100) 118 (49).
4.1.22. 5-(2-Hydroxy-3-methoxybenzylidene)-2-thioxodihydro-4,6(1H,5H)-
pyrimidinedione (22)
°
1
Yield: 0.90 g (95%); MP 257 C: H-NMR: (DMSO-d6, 400 MHz): δ 12.4 (s, 1H, NH),
12.3 (s, 1H, NH),9.9 (s, 1H,OH), 8.7 (s, 1H, CH aldehydic), 7.8 (d, 1H, J6,5 = 8.4 Hz, H-
6), 7.2 (d, 1H, J4,3 = 7.6 Hz,H-4), 6.8 (m, 1H, H-5), 3.8 (s, 3H, OCH3); EI-MS: m/z (rel.
int. %): 278 (M+, 41), 218 (9), 208 (15), 202 (100), 119 (8).
10

4.1.23. 5-[(5-Methyl-2-furyl)methylene]-2-thioxodihydro-4,6(1H,5H)pyrimidinedione
(23)
°
1
Yield: 0.89 g (93%); MP 235 C: H-NMR: (DMSO-d6, 300 MHz): δ 12.4 (s, 1H, NH),
12.3 (s, 1H, NH), 8.5 (d, 1H, J2,3 = 3.6 Hz, H-2), 7.9 (s, 1H, CH aldehydic), 6.7 (d, 1H,
J3,2 = 3.6 Hz, H-3), 1.8 (s, 3H, CH3); EI-MS: m/z (rel. int. %): 236 (M+, 100), 221 (63),
176 (47) 134 (35).
4.1.24. 5-(3,4-Dihydroxybenzylidene)-2-thioxodihydro-4,6(1H,5H)-pyrimidinedione (24)
°
1
Yield: 0.96 g (94%); MP 253 C: H-NMR: (DMSO-d6, 300 MHz):δ 12.3 (s, 2H, NH),
10.6 (s, 1H, OH), 9.6 (s, 1H,OH), 8.3 (s, 1H, H-2), 8.1 (s, 1H, CH aldehydic), 7.7 (d, 1H,
J6,5 = 7.2 Hz, H-6), 6.9 (d, 1H, J5,6 = 8.4 Hz, H-5); EI-MS: m/z (rel. int. %): 264 (M+,
100), 263 (85), 247 (59), 204 (32), 188 (23).
4.1.25. 5-(2-Hydroxy-5-methoxybenzylidene)-2-thioxodihydro-4,6(1H,5H)-
pyrimidinedione (25)
°
1
Yield: 0.89 g (93%); MP 249 C: H-NMR: (DMSO-d6, 300 MHz): δ 12.4 (s, 2H, NH),
11.5 (s, 1H, OH),8.2 (s, 1H, CH aldehydic), 7.9 (d, 1H, J3,4 = 8.2 Hz, H-6), 7.7 (s, 1H, H-
6), 7.5 (d, 1H, J4,3 = 8.4 Hz, H-5), 3.8 (s, 3H, OCH3); EI-MS: m/z (rel. int. %): 278 (M+,
90), 243 (10), 208 (40), 203 (100), 118 (49).
4.1.26. 2-Thioxo-5-(2,3,4-trihydroxybenzylidene)dihydro-4,6(1H,5H)-pyrimidinedione
(26)
°
1
Yield: 0.91 g (93%); MP 255 C: H-NMR: (DMSO-d6, 300 MHz): δ 12.5 (s, 2H, NH),
11.1 (s, 3H, OH), 8.8 (s, 1H, CH aldehydic), 7.5 (d, 1H, J6,5 = 8.7 Hz, H-6), 7.07 (d, 1H,
J5,6 = 8.4 Hz, H-2); EI-MS: m/z (rel. int. %): 280 (M+, 100), 260 (74), 234 (93), 176 (66),
134 (43).
4.1.27. 5-(1H-Indol-3-ylmethylene)-2-thioxodihydro-4,6(1H,5H)-pyrimidinedione (27)
°
1
Yield: 0.92 g (89%); MP 265 C: H-NMR: (DMSO-d6, 300 MHz): δ 12.9 (s, 1H, N’H),
12.2 (s, 1H, NH), 12.17 (s, 1H, NH),9.6 (s, 1H, H-2), 8.7 (s, 1H, CH aldehydic), 7.9 (d,
1H, J4,5 = 6.6 Hz, H-4), 7.6 (d, 1H, J7,6 = 5.7 Hz, H-7), (m, 2H, H-5/6); EI-MS: m/z (rel.
int. %): 271 (M+, 100), 270 (28), 196 (14), 172 (37), 168 (17).
11

4.2 Urease Inhibition Assay
Reaction mixtures comprising one unit of urease enzyme (Bacillus pasteurii) solution and
55 µL of buffers containing 100 mM urea were incubated with 5µL of test compounds
°
(1mM concentration) at 30 C for 15 min in 96-well plates. Urease activity was
determined by measuring ammonia production using the indophenol’s method.22
Momentarily, 45 µL each of phenol reagent and 70 (L of alkali reagent were added to
each well. The increasing absorbance at 630 nm was measured after 50 min, using a
micro-plate reader (Molecular Devices, USA). All reactions were performed in triplicate
in a final volume of 200 µL. The results (change in absorbance per min) were processed
by using the Soft-Max Pro s4.5.Software (Molecular Devices, USA).
Acknowledgements: The authors are thankful to the Organization for Prohibition of
Chemical Weapons (OPCW), The Netherlands (Project No. L/ ICA/ ICB/ 173681/ 12) for
financial support.
References
[1]
[2]
[3]
Mobley, H.; Island, M. D.; Hausinger, R. P. Microbiol. Rev. 1995, 59, 451-480.
Bayerdörffer, E.; Ottenjann, R. J. Gastroenterol. 1988, 23, 93-100.
Devesa, S. S.; Blot, W. J.; Fraumeni, J. F. Changing patterns in the incidence of
esophageal and gastric carcinoma in the United States, Cancer. 1998, 83, 2049-
2053.
[4]
[5]
[6]
Howson, C. P.; Hiyama, T.; Wynder, E. L. Epidemiol. Rev. 1986, 8, 1-27.
Parkin, D. M.; Bray, F. I.; Devesa, S. S. Eur. J. Cancer. 2001, 37, S4-S66.
López-Muñoz, F.; Ucha-Udabe, R.; Alamo, C. Neuropsychiatr. Dis. Treat. 2005,
1, 329-343.
[7]
Mobley, H.; Hausinger, R. Microbiol. Rev. 1989, 53, 85-108.
12

[8]
Rauf, A.; Ahmed, F.; Qureshi, A. M.; Khan, A.; Qadir, M. I.; Choudhary, M. I.;
Chohan, Z. H.; Youssoufid, M. H.; Haddad, T. B. J. Chin. Chem. Soc. 2011, 58,
528-537.
[9].
Dabholkar, V. V.; Ravi, T. D. J. Serbian Chem. Soc. 2010, 75, 1033-1040.
[10]. Kidwai, M.; Thakur, R.; Mohan, R. Acta. Chim. Slov. 2005, 52, 88-92.
[11]. Lee, J. H.; Lee, S.; Park, M.Y.; Myung, H. J. Virol. 2011, 8, 18. doi:
10.1186/1743-422X-8-18
[12]. Balas, V. I.; Verginadis, I. I.; Geromichalos, G. D.; Kourkoumelis, N.; Male, L.;
Hursthouse, M. B.; Repana, K. H.; Yiannaki, E.; Charalabopoulos, K.; Bakas, T.;
Hadjikakou, S. K. Eur. J. Med. Chem. 2011, 46, 2835-2844.
[13]. Brewer, A. D.; Minatelli, J. A.; Plowman, J.; Paull, K. D.; Narayanan, Biochem.
Pharmacol. 1985, 34, 2047-2050.
[14]. Yan, Q.; Cao, R.; Yi, W.; Yu, L.; Chen, Z.; Ma, L.; Song, H. Bioorg. Med. Chem.
Lett. 2009, 19, 4055-4058.
[15]. Vijaya Laxmi, S.; Thirupathi Reddy, Y.; Suresh Kuarm, B.; Narsimha Reddy, P.;
Crooks, P. A.; Rajitha, B. Bioorganic & Med. Chem. Letters, 2011, 21, 4329-
4331.
[16]. Thirupathi Reddy, Y.; Sekhar, K. R.; Nidhish Sasi, P.; Narsimha Reddy.;
Freeman, M. L.; Crooks, P. A. Bioorganic & Med. Chem. Letters. 2010, 20, 600-
602
[17]. Narsimha Reddy Penthala, Purushothama Rao Ponugoti, Vinod Kasam, Peter A.
Crooks. Bioorg. Med. Chem. Lett. 2013, 23, 1442-1446.
[18]. Chenguang, F.; Clay, M. D.; Deyholos, M. K.; John, C. Bioorg. Med. Chem.
2010, 18, 2141-2151
[19]. Orhan, D. D.; Kupeli, E.; Yesilada, E.; Ergun, F. J. Biosci. 2006, 61, 26-30.
[20]. Madadi, N. R.; Penthala, N. R.; Janganati, V.; Peter, A. Crooks. Bioorg. Med.
Chem. Lett. 2014, 24, 601-603.
13

[21]. a) Khan, K. M.; Ali, M.; Wadood, A.; Khan, M.; Lodhi, M. A.; Perveen, S.;
Choudhary, M. I.; Voelter, W. J. Mol. Graph. Model. 2011, 30, 153-156. b) Khan,
K. M.; Ali, M.; Ahmad, A.; Amyn, A.; Karim, A.; Khan, M.; Perveen, S. J.
Chem. Soc. Pak. 2013, 35, 890-893. c) Khan, K. M.; Khan, M.; Karim, A.; Taha,
M.; Ambreen, N.; Gojayev, A.; Perveen, S.; Choudhary, M. I. J. Chem. Soc. Pak.
2013, 35, 495-498. d) Khan, K. M.; Khan, M.; Ali, M.; Taha, M.; Hameed, A.;
Ali, S.; Perveen, S.; Choudhary, M. I. Med. Chem. 2011, 7, 231-236. e) Khan, K.
M.; Ali, M.; Ajaz, A.; Perveen, S.; Choudhary, M. I.; Atta-ur-Rahman Lett. Drug
Des. Discov. 2008, 5, 286-291. f) Khan, K. M.; Ali, M.; Khan, M.; Taha, M.;
Perveen, S. Lett, Org. Chem. 2011, 8, 28-32. g) Khan, K. M.; Ali, M.; Farooqui,
T. A.; Khan, M.; Taha, M.; Perveen, S. J. Chem. Soc. Pak. 2009, 31, 823-828.
[22]. Weatherburn, M. Anal. Chem. 1967, 39, 971-974
Scheme and Tables captions
Scheme; General Scheme for the synthesis of thiobarbituric acid derivatives 1-27
Table-1; Synthesis of thiobarbituric acid derivatives 1-27
Table-2; Urease activity of thiobabituric acid derivatives 1-27
14

Scheme 1
15

Table-1:
S.
Yield S.
No.
Yield S.
No.
Yield
R
R
R
No.
93%
93%
1
2
3
10
19
93%
92%
11
20
92%
91%
93%
90%
OH
12
21
93%
93%
88%
95%
93%
94%
4
5
13
22
93%
92%
14
23
93%
93%
6
7
8
15
24
16
17
25
26
95%
93%
91%
89%
93%
93%
H
N
92%
91%
89%
9
18
27
Table-2:
16

Compound No. IC50 SEMa(µ/M) Compound No. IC50 SEMa (µ/M)
46.5 0.56
47.3 0.62
21.4 1.04
21.5 0.61
18.1 0.52
62.8 1.71
16.0 0.45
16.0 0.22
43.3 1.06
22.8 0.32
14.3 0.27
42.5 1.3
50.3 0.81
32.3 0.62
6.7 0.27
1
14
15
16
17
18
19
20
21
22
23
24
25
26
27
2
3
10.6 0.17
58.5 1.28
42.1 1.91
29.4 0.59
32.1 0.34
19.2 0.29
18.2 0.76
1.61 0.18
32.7 0.82
25.2 0.63
170.8 2.08
4
5
6
7
8
9
10
11
12
137.2 1.92
21 0.11
13
Thioureab
SEMa is the standard error of the mean, Thioureab standard inhibitor for antiurease activity
17

Synthesis and SAR Studies of Thiobarbituric Acid Derivatives as Potent Inhibitors
of Urease
Khalid Mohammed Khan,1* Fazal Rahim,2 Ajmal Khan,1 Muhammad Shabeer,2 Shafqat
Hussain,1 Wajid Rehman,2 Muhammad Taha,3,4 Momin Khan,6 Shahnaz Perveen,5 and M.
Iqbal Choudhary1
1H. E. J. Research Institute of Chemistry, International Center for Chemical and
Biological Sciences, University of Karachi, Karachi-75270, Pakistan
2Depatment of Chemistry, Hazara University, Mansehra, Pakistan
3Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA
(UiTM), Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor, Malaysia,
4Faculty of Applied Science UiTM, 40450 Shah Alam, Selangor, Malaysia,
5PCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi
75280, Pakistan
6Department of Chemistry, Abdul Wali Khan University Mardan, Mardan-23200,
Pakistan.
*Corresponding Author: khalid.khan@iccs.edu, hassaan2@super.net.pk, Tel.: 0092-21-34824910; Fax:
0092-21-34819018
________________________________________________________________________
______
A series of thiobarbituric acid derivatives 1-27 were synthesized and evaluated for their
urease inhibitory potential. Amazing results were obtained from the analysis of these
compounds 1-27. Compounds 5, 7, 8, 11, 16, 17, 22, 23 and 24 showed excellent urease
inhibition with IC50 values of 18.1 0.52, 16.0 0.45, 16.0 0.22, 14.3 0.27, 6.7 0.27,
10.6 0.17, 19.2 0.29, 18.2 0.76 and 1.61 0.18 µM respectively more potent than the
standard inhibitor thiourea having IC50 value of 21.8 0.11 µM. Two compounds 3 and 4
exhibit potent inhibition with IC50 value 21.4 1.04 and 21.5 0.61µM almost same to the
standard inhibitor. The rest of the compounds also showed good urease inhibition. The
structure activity relationship was established for these compounds.
18